Our primary focus is the therapy of hematologic malignancies with recombinant toxins, and in the study of the interaction of toxins with the patient's malignant and normal tissues. A second, partially overlapping focus is in the development of new treatment paradigms for hairy cell leukemia (HCL), both early and multiply relapsed. 1. Clinical development of anti-CD22 recombinant immunotoxins BL22 and HA22 for HCL. To make a recombinant immunotoxin, the binding domain of a bacterial toxin like Pseudomonas exotoxin (PE) is replaced with a ligand, either a growth factor or an Fv fragment of a monoclonal antibody (MAb), which binds to a tumor antigen. It binds and is internalized by malignant cells. Cell death results after the catalytic domain of the toxin enters the cytosol. BL22 contains an anti-CD22 Fv fused to truncated PE, and has lifesaving activity in chemoresistant hairy cell leukemia. We have reported CR rates of 47-61% and overall response rates (ORR) of 72-81%. Of 36 phase II patients, 11 (65%) of 17 achieving CR have remained in CR for 32-68 (median 53) months. Smaller spleen size was associated with excellent chance for response (ORR 95%), compared to patients with larger spleens or post-splenectomy, arguing for initiating immunotoxin therapy earlier in the course of disease. The most common serious toxicity was a completely reversible hemolytic uremic syndrome (HUS), and after recovering, patients have maintained normal renal function for a median of 84 months without any evidence of recurrent HUS. Thus, BL22 is highly active producing durable remissions in chemoresistant HCL, particularly in patients with limited disease burden. To improve the targeting of chronic lymphocytic leukemia (CLL), which has lower CD22 density than HCL (median 1250 vs 44,000/cell), BL22 was mutated and a high-affinity recombinant immunotoxin produced, called HA22 or CAT-8015. A phase I trial of HA22 in HCL was recently completed, in which 28 patients were treated, and 10 additional patients were recently treated at the MTD. The ORR in the first 28 was 79%, and of the 25 patients in the upper 5 dose levels, the CR rate was 48%. No severe toxicities were observed on this trial and only 2 episodes of grade 2 HUS were observed. Thus HA22 was safe and highly effective in multiply relapsed HCL, and new trials are being planned. Clinical testing in CLL and non-Hodgkins lymphoma is underway via a multicenter phase I trial. A phase I trial in childhood acute lymphoblastic leukemia is also underway in collaboration with Alan Wayne, and CRs are already reported. 2. Clinical development of anti-CD25 recombinant immunotoxin LMB-2 for CD25+ CLL, HCL and cutaneous T-cell lymphoma (CTCL). LMB-2 is effective in CD25+ HCL, and is potentially life-saving for patients who have had HUS with BL22 and cannot receive additional anti-CD22 immunotoxin. Of 2 such patient treated on a phase II trial of LMB-2 in HCL, durable PR and CRs were achieved. Activity is also observed as a single agent in CLL and CTCL. Although nearly all patients with CTCL show benefit from LMB-2, retreatment is limited by immunogenicity, possibly due to prior infection with Pseudomonas aeruginosa. Thus LMB-2 may be useful for CTCL patients without prior Pseudomonas exposure. 3. Use of fludarabine and cyclophosphamide (FC) to both prevent immunogenicity and improve response to LMB-2 in patients with adult T-cell leukemia (ATL). To prevent immunogenicity and progression between cycles of LMB-2, patients with ATL, an aggressive T-cell leukemia, are being treated with FC chemotherapy prior to initiating cycles of FC followed by LMB-2. Our hypothesis is that intratumoral (interstitial) soluble CD25 (sCD25) may limit distribution of LMB-2 to rapidly growing tumor cells. So far, a CR was achieved by this approach. Biopsies are also taken to quantify intratumoral sCD25 in the patient tumors. Finally, an animal model was developed and showed synergy between LMB-2 and chemotherapy in the treatment of human CD25+ tumors. 4. New treatment strategies for early hairy cell leukemia: Because evidence of cure for HCL by purine analogs like cladribine is lacking, we have questioned the greater than 20 year standard practice of using cladribine alone as 1st or 2nd line treatment. To determine if the anti-CD20 Mab rituximab can eradicate minimal residual disease (MRD) in HCL and potentially result in cure, we have initiated a randomized trial in which patients receive cladribine combined with rituximab either up front or delayed at least 6 months. This trial is accruing we and is expected to enroll 150 patients, and should determine whether the early treatment of HCL should be changed. For multiply relapsed patients, most ineligible for immunotoxin, a randomized trial of bendamustine + rituximab vs pentostatin + rituximab is underway to define the activity of each regimen in HCL and to select the best regimen. These trials support our immunotoxin trials by creating a larger patient base and allowing assessment of many of the secondary endpoints. 5. Molecular characterization of HCL with respect to immunoglobulin rearrangements, and assessment of MRD. Discovery and characterization of a new poor-prognosis variant of HCL expressing the VH4-34 immunoglobulin rearrangement. Using the surface IgG displayed on the hairy cells, we have developed a novel sequence-specific PCR test for HCL, able to detect 1 HCL cell in 1,000,000 normal. This is over 10-times more sensitive than flow cytometry and is by far the most sensitive test yet developed for MRD in HCL. We are prospectively cloning surface IgG in patients treated with immunotoxin to determine if and when HCL is eradicated. We are also constructing a large database of sequences of surface IgG expressed by HCL cells. We find significant differences in expressed IgG between patients with HCL and the poor prognosis variant, HCLv, with respect to both gene usage and mutation frequency. A new variant expressing VH4-34 was described which has particularly poor response to cladribine alone and poor overall survival from diagnosis, but is sensitive to recombinant immunotoxin. 6. Investigation of the cause of HUS in patients treated with BL22 or HA22. Patients are being prospectively studied to detect markers for this complication of BL22 and HA22. The excretion of BL22, HA22 and LMB-2 was studied in the urine of patients and animals. Patients are being assayed prospectively for ADAMTS-13 to determine of diminished cleavage of ultralarge multimers of von Willebrands factor is associated with HUS from BL22/HA22. Finally, patients treated with HA22 are being prospectively studied with aspirin to determine if this can block immunotoxin-induced HUS. 7. Investigation of tumor markers in patients with B-cell malignancies, including soluble CD22. We found that soluble CD22 (sCD22) could be detected in the serum and its level correlates closely with overall disease burden in both HCL and CLL. sCD25 and sCD22 are being studied in HCL patients after treatment with cladribine, rituximab, or immunotoxins to correlate with other measures of overall disease burden, including flow cytometry, cat scan, and bone marrow biopsy. In a recently published study, a sCD22 level of less than 2 ng/ml strongly correlated with CR. Our hypothesis is that these tumor markers may allow patients to be managed more accurately without invasive procedures like bone marrow biopsy, to allow more accurate estimation of disease-free survival after treatment, and to predict early relapse.